Synergistic Effects of Stirring and Aeration Rate on Carotenoid Production in Yeast Rhodotorula toruloides CCT 7815 Envisioning Their Application as Soap Additives

Abstract

The production of carotenoids by microbial organisms has gained significant interest due to the growing demand for natural products. Among the non-model oleaginous red yeasts, Rhodotorula toruloides stands out as an appealing host for natural carotenoid production. R. toruloides possesses the natural ability to metabolize a wide range of substrates, including lignocellulosic hydrolysates, and convert them into lipids and carotenoids. In this study, we focused on utilizing xylose, the main component of hemicellulose, as the major substrate for R. toruloides. We conducted a comprehensive kinetic evaluation to examine the impact of aeration and agitation on carotenoid production. Results in stirred-tank reactor demonstrated that under milder conditions (300 rpm and 0.5 vvm), R. toruloides accumulated over 70% of its cell mass as lipids. Furthermore, the highest carotenoid yields were achieved at high agitation rates (700 rpm), with carotenoid levels reaching nearly 120 µg/mL. Several carotenoids were identified, including β-carotene, γ-carotene, torularhodin, and torulene, with β-carotene being the major carotenoid, accounting for up to 70% of the total carotenoid content. The carotenoid-rich extract produced by R. toruloides under evaluated conditions was successfully incorporated into soap formulations, demonstrating the addition of antioxidant properties. This work provides a comprehensive understanding of xylose conversion into natural carotenoids by R. toruloides, presenting a promising avenue for their application in cosmetics. Furthermore, this study highlights the potential of a renewable and cost-effective approach for carotenoid production in the soap industry.

1. Introduction

To achieve the transition towards a bioeconomy, it is necessary to develop innovative procedures for the sustainable production of natural pigments that have superior life cycle assessments and reduced energy requirements compared to synthetic colorants [1,2,3]. Recent advancements have brought attention to the utilization of microbial sources to generate carotenoids, a type of naturally occurring pigment found in various organisms such as plants, animals, macro and microalgae, fungi, and bacteria [4,5]. Carotenoids are categorized into two primary groups in nature: carotenes, which are hydrocarbons such as β-carotene and α-carotene, and xanthophylls, which are oxygenated carotenoid derivatives like torularhodin [6]. Despite the availability of well-known carotenes and xanthophylls in the market, the food industry faces a significant challenge in discovering and incorporating novel natural carotenoids with unique color and health-promoting biological properties [7].

Microbial carotenoids are synthesized by oleaginous red yeasts, bacteria, and microalgae, which can accumulate these pigments intracellularly [8,9]. The production of carotenoids by microorganisms has the advantage of not requiring land or other resources typically used for food production and is also not affected by climate or season [10]. Carotenoid accumulation can be stimulated in oleaginous red yeasts through the application of various stressors such as light intensity, temperature, nutrient availability, and culture conditions [5]. For instance, increasing the light intensity during cultivation can promote carotenoid biosynthesis in certain strains of red yeast [11]. Similarly, the optimization of the nutrient composition of the growth medium can also enhance carotenoid production [12].

Among the oleaginous microorganisms, Rhodotorula toruloides (R. toruloides), previously classified as Rhodosporidium toruloides, is a red, heterothallic, bipolar yeast that belongs to the Pucciniomycotina (basidiomycetes) group [13]. This yeast has recently emerged as a promising candidate for various biotechnological applications [14]. It is a natural producer of carotenoids, which impart a red color to the cells and possess potent antioxidant properties [15]. These compounds have significant industrial applications, with carotenoids finding use as colorants, antioxidants, or vitamin A precursors, and are therefore important for the food, feed, and pharmaceutical industries [16]. Due to the multifactorial influence on carotenoid biosynthesis by R. toruloides, encompassing factors such as strain variation, carbon sources, and cultivation conditions including agitation and aeration rates, it is critical to determine the suitable conditions for carotenoid production. Typically, preliminary studies utilize batch cultivation in flasks to identify such conditions, including agitation and aeration [17,18]. However, evaluating the impact of these parameters in shake flasks poses a significant challenge, primarily due to the uncontrollable nature of certain variables. Specifically, controlling the oxygen transfer rate (OTR) in shake flasks proves difficult due to the influence of various factors, such as the shaking speed, flask size and shape, culture volume, medium composition, and microorganism properties [19]. This variability in aeration conditions among different shake flasks poses a challenge in comparing the outcomes of various studies.

In addition, R. toruloides has the capacity to utilize various carbon and nitrogen sources, including lignocellulosic hydrolysates such as sugarcane bagasse, and can produce a diverse range of relevant compounds [20]. As hemicellulose, consisting mainly of polymerized five-carbon sugars (i.e., xylose and glucose), is the second most abundant fraction of lignocellulose after cellulose, it is imperative that a microorganism efficiently utilizes xylose to enhance the conversion of lignocellulosic materials into carotenoids [21]. Since xylose and glucose are carbon sources obtained from agro-industrial residues, such as sugarcane bagasse, they can be utilized for the biosynthesis of pigments in R. toruloides, thereby promoting the synthesis rate of carotenoids. Therefore, comprehending the effect of aeration and agitation rates on the mechanisms involving sugar consumption for the biosynthesis of carotenoids from xylose by R. toruloides is crucial to further enhance the yields, titers, and rates of this bioprocess, thereby increasing the economic feasibility of biotechnological processes in biorefineries.

As previously mentioned, the production of carotenoids by yeasts is influenced by various factors, including yeast strain, culture medium composition, and external physical factors such as light, temperature, aeration, osmotic stress, etc. [22]. Martins et al. [23] evaluated the production of carotenoids by two oleaginous red yeasts (R. toruloides and R. mucilaginosa) using sugar beet pulp hydrolysates. The authors revealed that R. toruloides produced the highest total carotenoid content (255 μg/g_dcw), whereas R. mucilaginosa produced the lowest amount of total carotenoids (81 μg/g_dcw). Saenge et al. [24] also revealed that the production of carotenoids by Rhodotorula can be further improved in a stirred-tank bioreactor with an improvement of the aeration rate from 0 to 2 vvm reaching 135.25 mg/L of total carotenoids.

In this study, a synthetic medium composed of xylose and glucose (in a 6:1 ratio), which was used to mimic the carbon source environment of sugarcane bagasse, was utilized to produce value-added carotenoids by R. toruloides. The goal was to explore the potential biorefinery applications for the valorization of sugarcane residues. For that, the effects of the volumetric oxygen transfer coefficient (k_La) and oxygen transfer rate (OTR) were first investigated. The culture conditions, including agitation and aeration rates, were optimized in a stirred-tank bioreactor. The present study also developed a batch process to enhance the production of specific carotenoids, namely β-carotene, γ-carotene, torulene, and torularhodin. Furthermore, the viability of cells and the properties of carotenoids (i.e., antioxidant activity) were also determined. Finally, to demonstrate the potential applications of the carotenoids-rich extract in cosmetic products, the pigments obtained in the fermentation was directly used as an additive incorporated in a soap formulation.

2. Materials and Methods

2.1. Determination of $k_{L}a$ and OTR

The volumetric oxygen transfer coefficient (${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$) was determined in a 7.5-L stirred-tank bioreactor Tecnal (TEC-BIO-7,5VI Tecnal, Brazil) operating with distilled water at 28 °C in the absence of microorganisms under different airflow rates and agitation speeds using the dynamic method previously described by Cerri et al. [25], taking into account the delay time. This involved measuring the concentration of dissolved oxygen in the fluid using an electrode (Mettler-Toledo InPro6000 series) following a change in the inlet gas (from N₂ to air) and recording the dissolved oxygen concentration values over time until saturation (100%) was reached. The ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ (s⁻¹) was determined using Equation (1):

$$-\mathrm{l}\mathrm{n}\left(1-\frac{{\mathrm{C}}_{{\mathrm{O}}_2}}{{\mathrm{C}}_{{\mathrm{O}}_2}^{\mathrm{*}}}\right)={\mathrm{k}}_{\mathrm{L}}\mathrm{a}.\mathrm{t}$$

(1)

In this expression, C_O2 and ${\mathrm{C}}_{{\mathrm{O}}_2}^{\mathrm{*}}$ are the concentration of dissolved oxygen in the liquid phase and at equilibrium conditions, respectively, and t is the time. The ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ values were obtained in triplicate, with N ranging from 300, 500, and 700 rpm and specific airflow rates of 0.5, 1.0, and 1.5 vvm, respectively.

Subsequently, the values of OTR (mmol.L⁻¹.s⁻¹) were calculated from the values of ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ and dissolved oxygen concentration as a comparison parameter using Equation (2):

$$\mathrm{O}\mathrm{T}\mathrm{R}={\mathrm{k}}_{\mathrm{L}}\mathrm{a}.\mathrm{H}.{\mathrm{P}}_{\mathrm{a}\mathrm{t}\mathrm{m}}.{\mathrm{x}}_{\mathrm{O}2}$$

(2)

where ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ is the volumetric oxygen transfer coefficient (s⁻¹), H is the Henrys law constant (mol.L⁻¹.Pa⁻¹), ${\mathrm{x}}_{\mathrm{O}2}$ is the oxygen molar fraction (0.23) in the gas inlet, and ${\mathrm{P}}_{\mathrm{a}\mathrm{t}\mathrm{m}}$ is the atmospheric pressure (Pa).

2.2. Strain and Inoculum

Rhodotorula toruloides (previously known as Rhodosporidium toruloides) CCT 7815 was acquired from the “Coleção de Culturas Tropicais” (Fundação André Tosello, Campinas, Brazil) and stored at −80 °C in 10% (v/v) glycerol. The pre-inoculum was prepared from frozen strain samples stored in Eppendorf tubes in YPD medium (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract) with 10% glycerol at a temperature of −15 °C. To prepare the pre-inoculum, a strain tube (1 mL) was thawed, and 9 mL of YPD medium was added to a Falcon tube at 28 °C and 200 rpm for 24 h. After that, the pre-inoculum production was started by cultivating a mixture of 10 mL of pre-inoculum and 90 mL of YPD medium in a 500 mL Erlenmeyer flask at 28 °C and 200 rpm for 24 h. Subsequently, 10 mL of this pre-inoculum was added to Erlenmeyer flasks (500 mL) containing 90 mL of YPD medium at 28 °C and 200 rpm for 24 h for cultivation. After this period, the cell suspension was centrifuged using an Eppendorf model 5804 R centrifuge at 1789× g, 4 °C for 15 min. The supernatant was discarded, and the cells were resuspended and diluted as needed in saline solution (0.9% NaCl) to reach an OD₆₀₀ (optical density at 600 nm) of 10.0 AU (absorbance units) for further inoculation in the production medium.

2.3. Production of Carotenoids Using a Stirred-Tank Bioreactor

The production of carotenoids using R. toruloides involved transferring the pre-inoculum to a 7.5-L stirred-tank bioreactor (Tecnal^®, model Tec-Bio-Flex, Piracicaba, SP, Brazil) equipped with a disc impeller, oxygen, and pH electrodes. The bioreactor contained 5 L of optimized cultivation medium composed of 70 g/L of carbon source (xylose and glucose in a mass ratio of 6:1), 1.5 g/L of MgSO₄·7H₂O, 2.0 g/L of yeast extract, 0.4 g/L of (NH₄)₂SO₄, 3.6 g/L of K₂SO₄, and 1% of trace element solution. This medium was selected to simulate the nutritional conditions of a hemicellulosic hydrolysate from sugarcane bagasse. Prior to use, the cultivation medium was sterilized at 121 °C for 30 min. The pH of the medium was adjusted to 6.0 at the start of the fermentation and remained constant throughout the process. The fermentation was conducted for 120 h at 28 °C with agitation at 300, 500, and 700 rpm and aeration at 0.5, 1.0, and 1.5 vvm (air volume/medium volume/min). Antifoam was added as needed. Samples were collected every 12 h to determine the concentration of carotenoids, sugars, dry cell weight, lipids, and xylitol/arabitol content.

2.4. Carotenoids-Rich Extract as Soap Additive

The preparation of soap was conducted following a modified procedure described by Mussagy et al. [26]. Initially, the commercial, neutral, and natural glycerine bar soap Memphis Ann Bow (Portão-RS, Brazil) was heated to 65 °C with moderated agitation until it became completely liquid. A predetermined amount of carotenoids extract was then added to the liquid soap and stirred for 10 min. Continuous mixing was maintained to ensure an efficient formulation. Upon achieving a dark orange semi-solid soap mass, the soap mixture was carefully poured into molds and left to solidify for a period of 12 h. Subsequently, the solid soaps were removed from the molds and stored in a dry and cool environment for a duration of two weeks to allow for further maturation. Following the two-week period, the solid soaps were analyzed, yielding a final concentration of 3.97 mg_carotenoids/g_soap. As a control specimen, a soap without the addition of carotenoids extract was prepared and evaluated alongside the modified soap samples.

2.5. Analytical Methods

2.5.1. Measurement of Sugars and Dry Biomass

The concentration of xylose and glucose in the supernatant was determined using high-performance liquid chromatography (HPLC) on a Dionex Ultimate 3000 system (ThermoScientific, Sunnyvale, CA, USA) equipped with an HPX-87H column (Biorad, Hercules, CA, USA) at a temperature of 50 °C. A 5 mmol/L solution of H₂SO₄ was used as the mobile phase with a flow rate of 0.6 mL/min. The sugars concentration (g/L) was determined according to the pre-established calibration curves obtained from pure xylose and glucose standards. The dry cell weight (DCW) for each sample (30 mL) was determined using the method previously described by Mussagy et al. [12]. After centrifuging the fermented broth at 2500× g for 10 min, the sample was washed twice with distilled water (1:2 w/v). Subsequently, the sample was dried in petri dishes at 100 °C for 24 h and weighed using an analytical balance (Shimadzu, model AUY220, Sao Paulo, SP, Brazil) to obtain the DCW.

2.5.2. Extraction and Quantification of Lipids

To quantify the lipid content of the R. toruloides biomass, we employed a modified version of the Bligh and Dyer method [27]. Initially, the cells were harvested by centrifugation (2000× g for 5 min at 25 °C), washed, and dried until a constant weight was achieved. The weight of the dry pellet was recorded using an analytical balance. Next, the dry pellet was suspended in 50 mL of a mixture containing chloroform/methanol (2:1 v/v) and vortexed for 5 min at at 25 °C. Subsequently, 10 mL of a 2 mol/L NaCl aqueous solution was added, and the mixture was centrifuged at 2000× g for 5 min at 25 °C to separate the aqueous and organic phases. The organic phase was then transferred to a rotary evaporator flask, dried under vacuum LR-271C (Grieve, IL, USA) at 200 mbar for 20 min until the organic phase was completely evaporated, and the final weight of the flask was measured. The lipid content was evaluated in g/L for comparison purposes.

2.5.3. Quantification of Carotenoids

The extraction and quantification of carotenoids were carried out following the methodology proposed by Mussagy et al. [28]. In this method, R. toruloides cells were disrupted using glass beads with a diameter of 425 to 600 μm, and acetone was used as the extracting agent to recover the pigments. This extraction process was repeated until the biomass was colorless, indicating the complete recovery of carotenoids. The carotenoids recovered were collected and then quantified by reversed-phase high-performance liquid chromatography, RP-HPLC, using column chromatograph C18 Acclaim 120, 4.6 × 250 mm, and the acetone-water gradient was used as the mobile phase at a flow rate of 1.0 mL/min (0 to 5 min, 80% acetone; 5 to 15 min, 100% acetone; 15 to 20 min, 80% acetone). The carotenoid concentrations were calculated according to the pre-established calibration curves obtained from pure β-carotene, γ-carotene, torulene, and torularhodin.

2.5.4. Antioxidant Activity Analysis Using DPPH Scavenging Assay

The antioxidant activity of the carotenoid and ascorbic acid (used for comparison purposes) was determined using the methodology previously described by Canaan et al. [29]. Furthermore, the antioxidant capacities of the formulated carotenoid-rich soaps were assessed to evaluate their bioactive properties. The modified DPPH• free-radical-scavenging activity assay was employed to determine the antioxidant activity of each soap. A concentration of 40 μg/mL of DPPH• in methanol was used in this assay. In brief, 0.18 g of each carotenoid-rich soap sample containing approximately 3.97 mg_carotenoids/g_soap was solubilized in 1 mL of methanol. Methanolic solution containing soap-carotenoids (100 μL) was filtered and mixed with 1.90 mL of the DPPH• solution. The same procedure was followed for the soap control, utilizing methanol instead. After a 30-min incubation period, the absorbance at 518 nm (UA) was measured using a UV–vis spectrophotometer. The antioxidant activity (%) was then determined by calculating the percentage reduction of the DPPH• absorbance, following Equation (3):

$$\mathrm{A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{t} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)=\frac{{\mathrm{A}}_{\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}}-{\mathrm{A}}_{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}}_{\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}}}\times 100$$

(3)

where A_DPPH represents the absorbance (UA) at 518 nm of the initial DPPH• solution, and A_Sample corresponds to the absorbance measured after the addition of the carotenoid-rich soaps and control soap.

2.6. Statistical Analysis

Statistical analysis was conducted using Statistica^® software 8.0 (StatSoft, Inc., Tulsa, OK, USA) to perform analysis of variance on three replicates with Tukey’s HSD test. The significance level for statistical differences was set at p < 0.05.

3. Results

3.1. Influence of Oxygen Transfer

The initial step of this study involved investigating the influence of oxygen transfer to select the initial conditions for the growth of aerobic R. toruloides yeast in a 5 L stirred-tank bioreactor. To achieve this, we evaluated the impact of agitation speed and aeration on two critical parameters: the volumetric oxygen transfer coefficient (${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$) and the initial oxygen transfer rate (OTR). In fact, by understanding the relationship between these parameters and efficient oxygen transfer, we can select the cultivation conditions for R. toruloides and enhance the biosynthesis of intracellular carotenoids. To do so, the ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ and OTR values were obtained in triplicate using distilled water at 28 °C, with an N range of 300 to 700 rpm, and at specific airflow rates of 0.5, 1.0, and 1.5 vvm (Figure 1).

Figure 1A illustrates the variation in ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ values, which ranged from 0.0086 to 0.066 h⁻¹. The results indicated that increasing the agitation speed (N) from 300 to 700 rpm had a positive effect on ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ values, with the highest value obtained at N = 1.5 vvm and 700 rpm (${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ = 0.066 h⁻¹).

3.2. Effect of Aeration and Agitation Rates on Carotenoids Production

The effect of the aeration rate and agitation speed on biomass and carotenoids production is shown in Figure 2.

Figure 2 illustrates that increasing the aeration rate from 0.5 to 1.5 vvm during a 120-h cultivation period resulted in an increase in biomass at impeller speeds of 300, 500, or 700 rpm. In addition, at agitation speeds of 300, 500 and 700 rpm, the biomass concentration increased with an increase in aeration rate from 0.5 to 1.5 vvm. The highest biomass concentration of 41 g/L was achieved in a culture that was grown under the highest aeration rate (1.5 vvm) and highest impeller speed (700 rpm), as indicated in Figure 2I. These results indicate that the increase in biomass was due to better air supply to the cells, which is particularly important for high biomass concentrations, since R. toruloides is a strictly aerobic microorganism. Therefore, high aeration rates combined with high impeller speeds improved the growth of the microorganism.

To evaluate the carotenoid profiles produced under various aeration and agitation conditions, Figure 3 displays the composition of carotenoids obtained from the bioreactor. The identification of the carotenoid profiles was based on a combination of information obtained from chromatographic and UV-vis features.

In Figure 3, the yeast R. toruloides produced four different carotenoids: β-carotene, γ-carotene, torulene, and torularhodin. The major carotenoid produced at 0.5 vvm and 300 rpm was β-carotene, accounting for approximately 70% of the total. Torularhodin and torulene followed in lower proportions. Because good conversion rates were obtained at 1.5 vvm, we decided to evaluate cell viability only under this condition. The effect of agitation rate on cell viability is shown in Figure 4.

As illustrated in Figure 4, the impact of agitation rate on cell concentration was investigated. At an agitation rate of 300 vvm, the total cell concentration was 9.92 × 10⁶/mL at time 0 h, and after 120 h (Figure 4A), a notable increase of 9.13 × 10⁷/mL in cell concentration was observed, with 28% of the cells being non-viable and 72% being viable. Similarly, at an agitation rate of 500 vvm, the initial cell concentration at time 0 h was 1.77 × 10⁷/mL, and a substantial increase of 9.91 × 10⁷/mL was observed after 120 h (Figure 4B). The percentage of non-viable and viable cells were 30% and 70%, respectively. Likewise, at an agitation rate of 700 rpm, the initial cell concentration at time 0 h was 2.11 × 10⁷/mL, and after 120 h (Figure 4C), a significant increase of 1.02 × 10⁸/mL in cell concentration was observed. The percentage of non-viable and viable cells were 30% and 70%, respectively.

3.3. Kinetics of Carotenoids Bioproduction

The next step of this work consisted of evaluating the carbon source consumption, biomass and lipids production, and total carotenoid yields of R. toruloides cultivated in the stirred-tank bioreactor at 28 °C for 120 h under CO₂ concentrations (0.05%) and O₂ (20%), in order to infer if the simultaneous consumption of sugars (xylose and glucose) would benefit the formation of bioproducts and provide some parameters to identify potential biotechnological applications of the yeast R. toruloides (Figure 5). For that, only the two-conditions were evaluated (500 and 700 rpm at 1 vvm). It is important to note that during our experimentation, we observed that both the 1 vvm and 1.5 vvm aeration conditions exhibited a similar trend in microbial carotenoid production at different agitation rates. Given these observations, we made the decision to focus our fermentation kinetics study on the 1 vvm aeration condition. This choice was made to streamline our investigation and concentrate on the kinetics of carotenoid production while minimizing the complexity introduced by evaluating multiple aeration levels.

Figure 5A,B displays the kinetics of carbon consumption, changes in metabolite contents in cell mass, as well as the kinetics of cell mass and metabolite production during the cultivation of R. toruloides. Yeast was cultivated on a mixture of carbon sources, xylose (60 g/L), and glucose (10 g/L), and both sugars were assimilated sequentially with glucose being consumed first, resulting in a diauxic growth pattern, as shown in Figure 5. Glucose consumption was observed to be very rapid, initiated at 9 h—not affected by the high availability of xylose— and with complete consumption within 40 h.

3.4. Antioxidant Activities of Ascorbic Acid and Carotenoid Extract

Generally, carotenoid-producing red yeast R. toruloides synthesize four types of carotenoids (β-carotene, γ-carotene, torulene, and torularhodin) and produce carotenoids at more than 120 µg/mL using xylose and glucose as a carbon source (ratio 6:1), at 28 °C, 700 rpm, and 1 vvm for 120 h. However, the overall production of carotenoids has been significantly influenced according to the various growth conditions used. To determine the antioxidant effect of carotenoids produced by R. toruloides, its free radical-scavenging capacity was measured with 2,2-diphenyl-1-picrylhydrazyl (DPPH) methanolic solution and compared with ascorbic acid, as depicted in Figure 6. Both ascorbic acid and carotenoids extract showed an antioxidative effect.

3.5. Carotenoids-Rich Extract as Soap Additive

To showcase the antioxidant properties of the carotenoids-rich extract obtained from R. toruloides, this extract was incorporated as an additive in the production of cosmetic soaps. The specific extract utilized in these products was the end-product obtained from the fermentation process using xylose and glucose as carbon sources (in a 6:1 ratio) at 28 °C, 700 rpm, and 1 vvm for 120 h. Notably, the carotenoid soap was prepared by directly including the final carotenoid extract as an additive, without undergoing any additional refining or purification procedures (Figure 7).

A comparison between the two soap formulations reveals that the carotenoid soap exhibits a significantly higher antioxidant activity, approximately six times greater than the control soap. The control soap, formulated with base ingredients containing oils known to possess antioxidant properties as confirmed by the DPPH assay, displayed an antioxidant activity level of approximately 10%. In contrast, the incorporation of carotenoid extracts into the soap formulation resulted in a substantial increase in antioxidant activity, showing a six-fold enhancement compared to the control soap.

4. Discussion

Agitation plays a crucial role in promoting oxygen transfer. Specifically, the use of radial flow impellers (Rushton turbines) was found to enhance fluid circulation within the bioreactor by altering the fluid flow profile, thus facilitating air bubble dispersion and improving mixing efficiency. Moreover, the ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ values obtained at high airflow rates (1.0 and 1.5 vvm) were consistent with those reported in previous studies using Rushton turbines [30], highlighting the positive impact of increased airflow on ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ values. Despite the positive effect of both N and air on the volumetric oxygen transfer coefficient, the agitation speed exhibited a higher effect than the specific airflow rate, a behavior consistent with previous observations available in literature [30]. The production of carotenoids by aerobic R. toruloides is influenced not only by agitation speed but also by the dissolved oxygen level. These parameters are crucial for determining the efficiency of oxygen transfer and are widely employed as aeration and agitation controls in aerobic processes [31]. To analyze the impact of the initial oxygen transfer rate (OTR), the same conditions used for evaluating k_La were applied. As shown in Figure 1B, the maximum OTR values obtained at agitation rates ranging from 300 to 700 rpm were as expected; the OTR values increased with agitation and aeration rate increase. The highest OTR values were found at the maximum tested speed (700 rpm), i.e., at 700 rpm; the maximum OTR values were 1.66, 4.63, and 7.84 × 10² mmol L⁻¹ s⁻¹ for 0.5, 1.0, and 1.5 vvm, respectively. The initial screening results revealed that increasing the agitation rate (to 700 rpm) and aeration (to 1.5 vvm) resulted in a significant increase in both the ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ and OTR values. This finding indicates that efficient oxygen transfer to the system resulted in increased uptake by the aerobic yeast R. toruloides. To further investigate the potential impact of ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ and OTR on biomass and carotenoid production, the next step of this study involved evaluating these outcomes under the evaluated conditions.

The combined effects of aeration and agitation under the evaluated conditions improve the mixing of the fermentation broth, which in turn maintains a concentration gradient between the interior and exterior of the yeast cells. This gradient facilitates the diffusion of nutrients into the cells while simultaneously aiding in the removal of gases and favors the mass transfer of substrate, product, and oxygen, and the carotenogenic metabolic pathways. In previous studies, Davoli et al. [32] demonstrated that increasing aeration rates enhances yeast carotenogenesis. R. glutinis yeast was found to produce approximately 45% more carotenoids when exposed to higher aeration rates. Similarly, Malisorn and Suntornsuk [33] reported that increasing the dissolved oxygen concentration in the medium from 60% to 80% in a 3-L stirred-tank bioreactor resulted in a positive influence on carotenoid production by R. glutinis (strain DM28). The maximum β-carotene production reached 0.21 mg/L after 24 h of fermentation. Additionally, Mussagy et al. [12] revealed that increasing the agitation rate (from 150 rpm to 350 rpm) and adding a further aeration source (1 vvm of air) in the cultivation of R. glutinis led to significant increases in the production of β-carotene (from 220.93 to 297.84 µg/mL), torularhodin (from 252.01 to 286.06 µg/mL), and torulene (from 28.20 to 37.35 µg/mL). Furthermore, Yamane et al. [34] achieved high rates of astaxanthin production by Phaffia rhodozyma when exposed to high oxygen concentrations. Regarding the influence of the agitation rate on the production of specific carotenoids by R. toruloides, we observed that 40% torularhodin production was achieved at 300 rpm, whereas torulene achieved almost similar production titers at 300, 500, and 700 rpm, revealing the low influence of agitation on the conversion of this carotenoid. This result is quite impressive because it demonstrates that there is no significant relationship between torulene composition and agitation speed. On the contrary, β-carotene exhibited high production rates at low agitation speed, following the trend of 300 rpm > 500 rpm > 700 rpm in all agitation speeds tested.

To obtain further information on the effect of agitation speed on the cell viability of R. toruloides, the next step in this work involved evaluating live/dead cell viability at agitation speeds of 300, 500, and 700 rpm.

R. toruloides is a highly promising oleaginous microorganism that can synthesize and store lipids within its cells to an impressive extent, with more than 50 wt% lipid content in its dry cell mass when using glucose or xylose as a carbon source [14,21,22]. In this study, the microorganism accumulated up to 9.97 g/L of lipids, which accounted for approximately 45% of its total biomass, under high aeration and agitation cultivation conditions. Under milder conditions (300 rpm and 0.5 vvm), the lipid accumulation reached approximately 32%. The results of this study indicate that a simple adjustment of the aeration and agitation conditions can significantly increase the production of carotenoids by R. toruloides, as well as facilitate the accumulation of other valuable by-products, such as lipids. The optimal cultivation medium was validated in a 5-L stirred-tank bioreactor, demonstrating that the culture media composition and aeration/agitation conditions are advantageous to produce carotenoids and lipid accumulation, as evidenced by the lipid content in the dry cell mass.

The results depicted in Figure 2 also demonstrate that in all culture systems, the biomass concentration increased rapidly during the first 72–120 h of cultivation (exponential growth). During this period, the carotenoid content also increased. Figure 2F, for instance, indicates that carotenoid production occurred in two phases. In the first phase (0–36 h of cultivation, referred to as the “growth phase”), only the biomass of the microorganism increased. In the second phase (after 36 h of cultivation, referred to as the “production phase”), both biomass increased and carotenoid biosynthesis occurred. In cultures subjected to an agitation speed of 300 rpm, the augmentation of the aeration rate from 0.5 to 1.0 vvm improved the carotenoid concentration in 48%, as depicted in Figure 2A,B. Nevertheless, as biomass production followed a similar trend, increasing agitation rates from 300 to 700 rpm and aeration rates from 0.5 to 1.5 vvm led to a carotenoid production of approximately 120 µg/mL obtained in a culture grown at an agitation speed of 700 rpm and aeration rate of 1.5 vvm after 120 h (Figure 2I). Concerning the effect of agitation speed on the cell viability of R. toruloides, the results suggest that a higher agitation rate can lead to a substantial increase in cell concentration, with a higher percentage of viable cells (70% in all conditions). In particular, the agitation rates of 500 vvm and 700 rpm produced a more considerable increase in cell concentration compared to an agitation rate of 300 vvm, revealing that agitation is crucial for cell growth but not necessary to produce some specific carotenoids (i.e., torulene).

Under low aeration conditions, R. toruloides may not efficiently convert β-carotene to other carotenoids such as torularhodin or torulene [35]. This is because the oxidation of the β-carotene molecule is required for the biosynthesis of the xanthophyll torularhodin, which may not occur effectively under low aeration. Increasing the aeration to 1.0 and 1.5 vvm was found to significantly improve the conversion of β-carotene to torulene and torularhodin. For instance, at 1.0 vvm, torulene production reached almost 35% at 700 rpm, whereas the highest content of torularhodin (approximately 35%) was obtained at 1.5 vvm and 300 rpm, indicating the successful conversion of β-carotene to torularhodin at high aeration rates.

Similar results were reported by Mussagy et al. [36] regarding the conversion of β-carotene to astaxanthin. The authors showed that increasing the aeration rate to 1 vvm was advantageous for the biosynthesis and conversion of β-carotene into astaxanthin. This was primarily due to the improved mass transfer of the substrate, which facilitated the specific metabolic pathways involved in the process. In general, the results obtained showed that the highest aeration rate (1.5 vvm) plays a critical role in the biosynthesis of carotenoids, particularly in the conversion of β-carotene to torularhodin and torulene. Under low aeration rates, the conversion of β-carotene to torularhodin may not occur efficiently, which suggests that higher aeration rates are required to produce these high-added value carotenoids.

Regarding bioproduct formation, it is important to note that the presence of excess xylose, as compared to glucose, is necessary for xylitol accumulation, as it upregulates the expression of xylose reductase and xylitol dehydrogenase. Note that efficient xylose consumption is also a prerequisite for the extracellular D-arabitol production by R. toruloides [37]. Our study demonstrated that increasing the agitation rates led to a higher consumption of xylose, which promoted the synthesis of xylitol. This led to a yield of 4.2 g/L, which was comparable to the yield obtained for D-arabitol. Umai et al. [38] also investigated the production of xylitol using R. toruloides NCIM 3547 with xylose-enriched hydrolysate. Their study resulted in yields of 4.73 g/L of xylitol after 168 h of fermentation under microaerophilic conditions. Similarly, Jagtap and Rao [37] evaluated the production of D-arabitol from xylose as the primary carbon source using R. toruloides IFO0880. Their study resulted in the production of 22, 32, and 49 g/L of D-arabitol from 70, 105, and 150 g/L xylose, respectively.

For glucose consumption, a similar observation was made for R. toruloides NCIM 3547 when a water hyacinth hydrolysate was used as the medium for a dual-sugar fermentation [38]. In this case, glucose consumption was initiated rapidly at 9 h and not affected by the availability of xylose. Xylose consumption, on the other hand, was somewhat repressed by the presence of glucose, and a rapid depletion of xylose occurred only after glucose levels were reduced to low levels. Our study showed similar results, with glucose being depleted after 40 h and xylose after 120 h (Figure 5A,B). The results from Figure 5A,B also indicate that biomass increases until the carbon sources are fully consumed, and carotenoid concentration is increased until glucose is depleted. A high accumulation of carotenoids was observed at 120 h, when both glucose and xylose were depleted, suggesting that the synthesis of carotenoids is influenced by sugar levels in the culture media, and carotenoid biosynthesis is improved when glucose is exhausted. The highest carotenoids yields were obtained at high agitation rates (700 rpm), achieving almost 120 µg/mL.

The scavenging activities of DPPH• exerted by carotenoids-rich extracts depicted in Figure 6 indicate the hydrogen-donating capability of carotenoids such as torularhodin (i.e., hydroxyl ending group) presented in the carotenoid-rich extract. The antioxidant activity of crude carotenoid extracts displayed the elevated percentages of inhibition of 100% and 60% at the highest concentration (46.6 and 93.2 mg/L, respectively) in the tested concentrations and was significantly different from the others (p < 0.05). As expected, the lower the concentration of the carotenoids-rich extract, the lower the antioxidant capacity of the extract, viz., at 5.85 mg/L of carotenoids, only 18% of inhibition was achieved. These results are in accordance with those obtained by Mussagy et al. [36]. In this work, the authors revealed that 100 mg/L of the carotenoids-rich extracts obtained by red yeast Phaffia rhodozyma inhibited 100% of DPPH radical. Chin Hu et al. [39] pointed out that carotenoids produced by Dunaliella salina, at 10 mg/mL of zeaxanthin, lutein, β-carotene and α-carotene, and α-tocopherol obtained from the carotenoids-rich extract, exhibited the scavenging abilities on DPPH radicals of 64, 26, 25, 24, 24, and 82% respectively. Moreover, when assessing the antioxidant potential of carotenoid extracts derived from R. toruloides in comparison to authentic antioxidant compounds such as ascorbic acid, the findings reveal that carotenoid-rich extracts exhibit nearly a 10% greater antioxidant capacity. This also indicates that the addition of carotenoid extracts significantly boosts the overall antioxidant activity of the final soap product, resulting in an increase of approximately 60%. The capacity of the carotenoids to act as free radical scavengers confirmed the intrinsic antioxidant nature of carotenoids produced by R. toruloides. As observed, the utilization of carotenoid extracts as additives in cosmetic products, such as soaps, offers a promising approach to enhance antioxidant activity, which contributes to the improved protection of consumers against skin damage caused by free radicals.

5. Conclusions

The production of carotenoids by R. toruloides CCT 7815 demonstrated enhanced growth rate and carotenoid production. We observed that a significant accumulation of carotenoids occurred when glucose was depleted, and the highest carotenoid yields were achieved at high agitation rates (700 rpm and 1.5 vvm), reaching nearly 110 µg/mL because of ${\mathrm{k}}_{\mathrm{L}}\mathrm{a}$ and OTR. The increase in agitation rates resulted in a higher consumption of xylose, leading to the synthesis of xylitol. Remarkably, the yield of xylitol (4.2 g/L) was comparable to that of D-arabitol. The carotenoids produced exhibited a high antioxidant capacity and were successfully incorporated as additives in soap formulations. This innovative approach offers an ecologically friendly and straightforward alternative to conventional soap additives, some of which still rely on the use of synthetic and potentially harmful chemicals.